解剖学报

解剖学报 ›› 2016, Vol. 47 ›› Issue (6): 756-762.doi: 10.16098/j.issn.0529-1356.2016.06.006

• 细胞和分子生物学 • 上一篇    下一篇

骨髓间充质干细胞抑制转化生长因子-β1诱导的肺泡Ⅱ型上皮细胞间质化

王鑫1 高俊玲1,2,3* 赵曼曼1 朱会兴1 田艳霞1 李冉1 江小华1 于雷1 田景瑞1 崔建忠4   

  1. 1. 华北理工大学基础医学院,河北 唐山 063000; 2. 河北省慢性疾病重点实验室，河北 唐山 063000; 3. 唐山市慢性病临床基础研究重点实验室，河北 唐山 063000; 4. 唐山市工人医院神经外科，河北 唐山 063000
  • 收稿日期:2016-04-25 修回日期:2016-06-29 出版日期:2016-12-06 发布日期:2016-12-06
  • 通讯作者: 高俊玲 E-mail:junlinggao@163.com
  • 基金资助:

    河北省自然科学基金面上项目

Inhibition of bone marrow mesenchymal stem cells on epithelial mesenchymal transformation induced by transforming growth factor-β1

WANG Xin1 GAO Jun-ling 1,2,3* ZHAO Man-man1 ZHU Hui-xing1 TIAN Yan-xia1 LI Ran1 JIANG Xiao-hua1 YU Lei1 TIAN Jing-rui1 CUI Jian-zhong4   

  1. 1.School of Basic Medical Sciences, North China University of Science and Technology, Hebei Tangshan 063000, China; 2.Hebei Key Laboratory for Chronic Diseases, Hebei Tangshan 063000, China; 3.Tangshan Key Laboratory for Preclinical and Basic Research on Chronic Diseases, Hebei Tangshan 063000, China; 4. Department of Neurosurgery, Tangshan Gongren Hospital; Hebei Tangshan 063000,China

  • Received:2016-04-25 Revised:2016-06-29 Online:2016-12-06 Published:2016-12-06
  • Contact: GAO Jun-ling E-mail:junlinggao@163.com

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摘要：

目的 探讨骨髓间充质干细胞（BMSCs）是否通过抑制肺泡Ⅱ型上皮细胞向肌成纤维细胞的转化，进而发挥其抗纤维化的作用及其机制。 方法 培养大鼠肺泡Ⅱ型上皮细胞系RLE-6TN细胞，用5μg/L的TGF-β1诱导分化，并分为对照组、转化生长因子（TGF）-β1刺激组、BMSCs干预组。采用倒置相差显微镜观察RLE-6TN细胞的形态变化；免疫荧光法检测E-钙黏蛋白（E-cad）、α-平滑肌肌动蛋白（α-SMA）的定位；Western blotting法检测E-cad、α-SMA、p-Smad3、Snail1蛋白的表达。 结果 在TGF-β1的诱导下，肺泡Ⅱ型上皮细胞逐渐向肌成纤维细胞发生形态改变，由上皮细胞的鹅卵石状变成间充质细胞的梭形或纺锤形，且伴随着上皮标志物E-cad表达降低，而间充质细胞标志物α-SMA表达上调；给予BMSCs干预后，间充质化有所抑制，表现于细胞形态趋于上皮型，且上皮标志物E-cad表达上调，间质标志物α-SMA表达减少。与对照组相比，TGF-β1刺激组p-Smad3、 Snail1蛋白表达上调。给予BMSCs干预后p-Smad3、Snail1蛋白表达显著降低。 结论 BMSCs可能通过阻断TGF-β1/Smad3信号转导通路，抑制肺泡Ⅱ型上皮细胞向肌成纤维细胞的转化，从而抑制上皮细胞-间充质转化的进程。

关键词: 骨髓间充质干细胞, 上皮细胞-间充质转化, 肺泡Ⅱ型上皮细胞, 免疫荧光, 免疫印迹法

Abstract:

Objective To explore whether bone marrow mesenchymal stem cells could inhibit epithelial-mesenchymal transition(EMT) of rat type Ⅱalveolar epithelial cells through inhibiting Smad3 phosphorylation, and play a role on anti pulmonary fibrosis. Methods Rat type Ⅱ alveolar epithelial cell line RLE-6TN cells were cultivated, induced by 5μg/L transforming growth factor(TGF)-β1 in vitro, and divided into normal control group. TGF-β1 induced group and BMSCs intervention group. Morphological changes of epithelial-mesenchymal transition in RLE-6TN cells were observed under an inverted phase contrast microscope. Immunofluorescence methods were used to determine the location of E-cadherin (E-cad) and α-smooth muscle actin（α-SMA). The expressions of E-cad, α-SMA, p-Smad3 and Snail1 were detected by Western blotting in each group. Results The process of EMT induced by TGF-β1 was accompanied by morphological alteration. RLE-6TN cells had an epithelial cobblestone-like morphology, a round or polygonal shape and exhibited very close cell-cell proximity, while after exposure to TGF-β1, the cells displayed spindle-shaped elongated fibroblast-like cells morphology. Concomitant with a down-regulation of the epithelial markers E-cad, the expression of interstitial cells markers α-SMA up-regulated. Similar morphological changes were noticeably suppressed in the BMSCs intervention group, and the BMSCs intervention group showed a reduction in TGF-β1-induced up-regulation of α-SMA, while a down-regulation of E-cad was observed. Compared with control group, the expression levels of p-Smad3 and Snail1 increased significantly after the stimulation of TGF-β1. After BMSCs intervention,the expressions of p-Smad3 and Snail1 significantly decrease compared to the TGF-β1 group. Conclusion BMSCs may block TGF-β1/Smad3 signal transduction pathway, and then inhibit epithelial mesenchymal transition of type Ⅱ alveolar epithelial cells.

Key words: Bone marrow mesenchymal stem cell, Epithelial-mesenchymal transition, Alveolar type Ⅱ cell, Immunofluorescence, Western blotting